Intraneuronal ?S aggregates (Lewy bodies and Lewy neurites) are pathological hallmarks of both familial and sporadic (?idiopathic?) PD as well as other ?synucleinopathies? including dementia with Lewy bodies, multiple system atrophy and even Alzheimer?s disease. Disease-modifying drugs for treating human synucleinopathies do not yet exist. This is due in part to a lack of compelling animal and cellular models that recapitulate the dynamic transition from physiological to non-physiological ?S states. Here we propose both new mouse models and efficient cellular drug screens based on our novel insights about ?S structure. The PI recently performed a mutagenesis screen for loss of 60/80/100 kDa putative ?S multimers that have now been observed by several labs as apparent native species. He identified key amino acids whose mutation lowers ?S60/80/100 levels and markedly perturbs cellular ?S homeostasis with toxic consequences. Importantly, the ?S variants we propose to study in this grant are basically ?amplifications? of the fPD/DLB-causing mutation E46K in KTKEGV repeat #4. They are made by inserting either 1 or 2 additional E46K-like mutations into flanking repeats #3 and 5. Unlike single ?S fPD point mutations that do not produce comprehensive and robust phenotypes in cell culture, the proposed ?amplification? strategy readily produces key features of pathological ?S ?in the dish?: increased ?S insolubility, progressive neurotoxicity and formation of round inclusions. The structural analogy to E46K will make our findings relevant for modeling & treating synucleinopathies. Our extensive preliminary data will be exploited in 3 major aims: 1. Novel ?S mouse models that express inclusion-prone ?SE35K+E46K (??S2K?) and ?SE35K+E46K+E61K (??S3K?) variants, plus ??SKLK?, another neurotoxic ?S motif-mutant. 2. Neuronal models of the toxic ?S variants. Special focus will be the characterization of the striking ?S inclusions that form in neuronal somata and neurites, determining their relationship, if any, to ?-sheet-rich ?S Lewy aggregates, and defining the inclusions as toxic or protective for the neuron. 3. Performing screens for factors (genes but principally small drug-like molecules) that can correct the protein dyshomeostasis that underlies this inclusion formation and neurotoxicity, with the goal of finding synucleinopathy-modifying drugs. All 3 Aims are based on detailed and technically enabling preliminary studies. We believe that the new research proposed herein will help overcome the lack of compelling rodent and cellular models to study early aspects of intraneuronal disease initiation in the pathogenesis of synucleinopathies. Our models will thus be complementary to approaches that focus on extracellular, non-cell-autonomous spreading models. The PI has a strong background in cell biology, biochemistry and neurodegenerative disease research and will conduct the work in his new, independent laboratory, in collaboration with experts on ?S mouse models (Silke Nuber), ?S drug screens (Susan Lindquist lab) and ?S biophysics (Tim Bartels). 1